Profiling of Eicosanoid Production in the Rat Hippocampus during Kainic Acid-induced Seizure

Abstract

Kainic acid (KA)-induced seizure in rat involves eicosanoid production in the brain, but their production mechanism and biological functions are poorly understood. We profiled the eicosanoid production during KA-induced seizure by a comprehensive lipidomics analysis using liquid chromatography-tandem mass spectrometry. Systemic KA administration caused production of large amounts of prostaglandin (PG) F2α and PGD2 in the hippocampus, with smaller amounts of other PGs and hydroxyeicosatetraenoic acids. The production was biphasic, which consisted of an initial burst in the first 30 min and a sustained late phase production. The initial phase was specific to the hippocampus and was blocked by intracerebroventricular administration of KA receptor antagonists. A selective cyclooxygenase (COX)-2 inhibitor, NS398, completely inhibited the initial phase productions, except for PGD2 and thromboxane B2, whose productions were also dependent on COX-1. These results suggest that KA signals directly stimulate the arachidonic acid cascade in the initial phase and that COX-1 and COX-2, both constitutively expressed at low levels, differentially contribute to PG productions. In the late phase, a sustained PG production in hippocampus appears due to the increased COX-2 levels even with a limited arachidonic acid supply. The present study demonstrates a dual phase regulatory mechanism of eicosanoid production during KA-induced seizure, providing a biochemical basis for understanding the biosynthesis and roles of eicosanoids in the brain. Kainic acid (KA)-induced seizure in rat involves eicosanoid production in the brain, but their production mechanism and biological functions are poorly understood. We profiled the eicosanoid production during KA-induced seizure by a comprehensive lipidomics analysis using liquid chromatography-tandem mass spectrometry. Systemic KA administration caused production of large amounts of prostaglandin (PG) F2α and PGD2 in the hippocampus, with smaller amounts of other PGs and hydroxyeicosatetraenoic acids. The production was biphasic, which consisted of an initial burst in the first 30 min and a sustained late phase production. The initial phase was specific to the hippocampus and was blocked by intracerebroventricular administration of KA receptor antagonists. A selective cyclooxygenase (COX)-2 inhibitor, NS398, completely inhibited the initial phase productions, except for PGD2 and thromboxane B2, whose productions were also dependent on COX-1. These results suggest that KA signals directly stimulate the arachidonic acid cascade in the initial phase and that COX-1 and COX-2, both constitutively expressed at low levels, differentially contribute to PG productions. In the late phase, a sustained PG production in hippocampus appears due to the increased COX-2 levels even with a limited arachidonic acid supply. The present study demonstrates a dual phase regulatory mechanism of eicosanoid production during KA-induced seizure, providing a biochemical basis for understanding the biosynthesis and roles of eicosanoids in the brain. The pathology of intractable temporal lobe epilepsy involves glutamate excitotoxicity: an activation of glutamate receptors by high concentration of glutamate causes neuronal damages in the brain (1Choi D.W. Neuron. 1988; 1: 623-634Abstract Full Text PDF PubMed Scopus (4212) Google Scholar). An excitotoxic amino acid, kainic acid (KA), 4The abbreviations used are: KA, kainic acid; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; cPLA2α, cytosolic phospholipase A2α; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; LT, leukotriene; mPGES-1, microsomal prostaglandin E synthase-1; PBS, phosphate-buffered saline; PG, prostaglandin; PGI synthase, prostaglandin I synthase; SRM, selected reaction monitoring; TxB2, thromboxane B2; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione. stimulates KA receptors, which are the members of non-N-methyl-d-aspartate ionotropic glutamate receptors. When systemically administrated to rats, KA induces a rapid and recurrent epileptic seizure, followed by a neuronal degeneration in specific brain areas, including hippocampus (2Sperk G. Prog. Neurobiol. 1994; 42: 1-32Crossref PubMed Scopus (631) Google Scholar). KA-induced seizure has been thus studied as one of the animal models of human temporal lobe epilepsy (3Ben-Ari Y. Neuroscience. 1985; 14: 375-403Crossref PubMed Scopus (1536) Google Scholar). Eicosanoids such as prostaglandins (PGs) and leukotrienes (LTs) are arachidonic acid-derived lipid mediators produced from membrane phospholipids, which exert diverse biological activities as intercellular signaling molecules through their cognate G protein-coupled receptors (4Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2105) Google Scholar, 5Moepps B. Fagni L. EMBO Rep. 2003; 4: 237-243Crossref PubMed Scopus (9) Google Scholar). Involvement of eicosanoids in the KA-induced seizure has been suggested; KA stimulates eicosanoid production in the rat brain (6Baran H. Heldt R. Hertting G. Brain Res. 1987; 404: 107-112Crossref PubMed Scopus (54) Google Scholar), and expression levels of enzymes such as cyclooxygenase (COX)-2 (7Chen J. Marsh T. Zhang J.S. Graham S.H. Neuroreport. 1995; 6: 245-248Crossref PubMed Scopus (110) Google Scholar, 8Manev H. Uz T. Qu T. Exp. Gerontol. 2000; 35: 1201-1209Crossref PubMed Scopus (36) Google Scholar, 9Matsuoka Y. Okazaki M. Kitamura Y. Takata K. Tooyama I. Kimura H. Taniguchi T. Brain Res. 1999; 836: 213-217Crossref PubMed Scopus (10) Google Scholar, 10Ciceri P. Zhang Y. Shaffer A.F. Leahy K.M. Woerner M.B. Smith W.G. Seibert K. Isakson P.C. J. Pharmacol. Exp. Ther. 2002; 302: 846-852Crossref PubMed Scopus (72) Google Scholar, 11Kim E.J. Lee J.E. Kwon K.J. Lee S.H. Moon C.H. Baik E.J. Brain Res. 2001; 908: 1-9Crossref PubMed Scopus (48) Google Scholar, 12Kawaguchi K. Hickey R.W. Rose M.E. Zhu L. Chen J. Graham S.H. Brain Res. 2005; 1050: 130-137Crossref PubMed Scopus (86) Google Scholar) and 5-lipoxygenase (8Manev H. Uz T. Qu T. Exp. Gerontol. 2000; 35: 1201-1209Crossref PubMed Scopus (36) Google Scholar) are affected by KA. However, the roles of eicosanoids as well as their production mechanisms are poorly understood. In the present study, we profiled the eicosanoid production in the rat hippocampus during KA-induced seizure. Taking advantages of the simultaneous quantification method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) (13Kita Y. Takahashi T. Uozumi N. Nallan L. Gelb M.H. Shimizu T. Biochem. Biophys. Res. Commun. 2005; 330: 898-906Crossref PubMed Scopus (33) Google Scholar, 14Kita Y. Takahashi T. Uozumi N. Shimizu T. Anal. Biochem. 2005; 342: 134-143Crossref PubMed Scopus (107) Google Scholar), we carried out a comprehensive analysis of lipid mediators in the rat hippocampal tissues. The time course of COX-2 up-regulation and the profile of lipid mediator production suggested a dual phase regulatory mechanism; a direct KA action may cause a transient burst PG production in the initial phase, whereas increased COX-2 levels mediate the late phase production under limited arachidonic acid supply. We also demonstrate a contribution of COX-1 in the production of PGD2 and TxB2 in the hippocampus, as well as a role of constitutively expressed COX-2, suggesting a diversity of PG production pathways in the brain. Animals—Three-week-old male Wistar rats, 3-day-old male rats, and embryonic day 18 rats (SLC, Hamamatsu, Japan) were used in the study. All animal studies were conducted in accordance with the Guidelines for Animal Research at the University of Tokyo and were approved by the Animal Ethics Committee of the University of Tokyo. Reagents—Kainic acid, NS398, and indomethacin were purchased from Wako (Osaka, Japan). UBP296, UBP301, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were from Tocris (Bristol, UK). [α-32P]CTP was from PerkinElmer Life Sciences. Rabbit polyclonal anti-cytosolic phospholipase A2α (cPLA2α) antibody was from Cell Signaling Technology (Beverly, MA), and rabbit polyclonal anti-COX-2 antibody was from Cayman Chemical (Ann Arbor, MI). Horseradish peroxidase-conjugated donkey anti-rabbit IgG was from Amersham Biosciences. All eicosanoid primary standards and deuterium-labeled eicosanoid internal standards for quantification were purchased from Cayman Chemical (Ann Arbor, MI). Drug Administration, Tissue Collection, and Lipid Extraction—Rats were euthanized at indicated periods after intraperitoneal KA administration (10 mg/kg, dissolved in saline). Hippocampi (70–80 mg) and cerebral cortex (70–100 mg) were dissected, immediately frozen under liquid nitrogen, and stored at –80 °C until use. The tissues were crushed to powder with an SK-100 mill (Tokken, Chiba, Japan) without thawing, and lipids were extracted with 1 ml of ethanol with a mixture of deuterium-labeled internal standards of eicosanoids. The extracts were further cleaned up with Oasis HLB extraction cartridges (30 mg, Waters, Milford, MA) as described (14Kita Y. Takahashi T. Uozumi N. Shimizu T. Anal. Biochem. 2005; 342: 134-143Crossref PubMed Scopus (107) Google Scholar). For the intracerebroventricular administration of KA receptor antagonists, CNQX, UBP296, and UBP301 were dissolved in phosphate-buffered saline (PBS)/Me2SO (95:5, v/v) at a concentration of 10 mm (50 nmol in 5 μl). 5 μl of the solutions was injected into both of the lateral ventricles by a 10-μl Hamilton microsyringe fitted with a 27-gauge × 3/4-inch length needle with the following coordinates: anterior/posterior =–1 mm behind bregma, medial/lateral = 1 mm, and an injection depth of 3.5 mm. For COX inhibitor administration, indomethacin and NS398 were dissolved in Me2SO (5 mg/ml) and intraperitoneally injected at a dose of 10 mg/kg, 30 min before KA administration. Quantification of Eicosanoids—Eicosanoids in the brain were quantified as described (14Kita Y. Takahashi T. Uozumi N. Shimizu T. Anal. Biochem. 2005; 342: 134-143Crossref PubMed Scopus (107) Google Scholar). Briefly, a TSQ-7000 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (Thermo Electron, Waltham, MA) was operated in negative-ESI and selected reaction monitoring (SRM) mode. A reversed phase high performance liquid chromatograph system, consisting of four Shimadzu (Kyoto, Japan) LC-10A pumps and a Shimadzu CTO-10 column oven, an electrically controlled 6-port switching valve (Valco, Houston, TX), and a 3033 HTS autosampler (Shiseido, Tokyo, Japan), was connected to the MS instrument and used for the rapid resolution of PGs with Shiseido Capcellpak C18 MGS3 (1 × 100 mm) column within 10 min. For the accurate quantification, an internal standard method was used. As internal standards, a mixture of deuterium-labeled eicosanoids was used. Automated peak detection, calibration, and calculation were carried out by the use of Xcalibur 1.2 software package (Thermo Electron). Reverse Transcription-PCR—For the preparation of first-strand cDNA templates from rat hippocampus, total RNA was isolated by Isogen reagent (Nippon Gene, Toyama, Japan), and mRNA was isolated by using a μMACS mRNA isolation kit (Miltenyi Biotec, Gladbach, Germany). Oligo(dT)-primed reverse transcriptions were performed with Superscript II enzyme (Invitrogen). Following primers were used for PCR: cPLA2α (forward, 5′-TTTGGCGATATGCTGGACACTCCTG-3′; reverse, 5′-GAGGGAAACAGAGCAACGAGATGGG-3′), COX-1 (forward, 5′-GGAGTCTCTCGCTCCAGTTTCCCCT-3′; reverse, 5′-GGAGACATAGGGGCAGGTCTTGGTG-3′), COX-2 (forward, 5′-ATGCAGCAAATCCTTGCTGTTCCAA-3′; reverse, 5′-CACTTGCGTTGATGGTGGCTGTCTT-3′), microsomal PGE synthase-1 (mPGES-1, forward, 5′-TTCCCTGGGTTTGGTGATGGAGAAC-3′; reverse, 5′-ACAGGCGAACTGGGCCAGAACATAG-3′), lipocalin-type PGD synthase (forward, 5′-CTCTTGGGATTTCCACAGACCCCAG-3′; reverse, 5′-ACTTATCCGGTTGGGGCAGGAAAAC-3′), PGI synthase (forward, 5′-ATGCCGCCAGCTTCCTTACCAGGAT-3′; reverse, 5′-GGCTGCATCAGGCCAAAGCCATATC-3′), and glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-GAAGGTCGGTGTGAACGGATTTGGC-3′; reverse, 5′-TTGTCATTGAGAGCAATGCCAGCCC-3′). Expected PCR product sizes were 2,088, 1,733, 1,701, 409, 511, 1,319, and 906 bp for cPLA2α, COX1, COX-2, mPGES-1, lipocalin-type PGD synthase, PGI synthase, and glyceraldehyde-3-phosphate dehydrogenase, respectively. PCR conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 30 cycles of 96 °C for 15 °C for 30 °C for 2 min cPLA2α, COX-2, and PGI 1 min mPGES-1, lipocalin-type PGD synthase, and glyceraldehyde-3-phosphate and at °C for 5 min. The were by RNA was in RNA were from tissues of at rats, and of which were the The RNA was membrane for and with were used for × to × of were by of cDNA in a using and RNA cDNA used and for cPLA2α, for and for COX-2 are on of and All the were selected within the of cDNA with were performed at °C for using 1 × of in a 5 × Japan), 5 × mm mm mm and mm under signals were with a Tokyo, Japan). were in a mm mm 10 mm a inhibitor mixture (1 of was to a and an membrane The membrane was blocked at °C with Japan) and for 1 at with rabbit polyclonal antibody for cPLA2α COX-2 in with the membrane was with donkey anti-rabbit IgG with for 1 signals were with an were carried out with package and of were PG in the after Systemic KA comprehensive analysis of eicosanoids in the rat hippocampal was carried out with a quantification 1 for hippocampal tissues 1 after intraperitoneal KA injection and from In system, of TxB2 of and were even in the rats and increased after KA administration and were hydroxyeicosatetraenoic acid of and were which increased after KA administration. deuterium-labeled internal standards, PGs were quantified up to after KA As in hippocampal produced and PGD2 as followed by TxB2, and in The PG levels a peak within 30 min to 1 after KA administration and to The production levels the levels at and sustained up to All PG a time course These results that hippocampal PG production after KA administration was an initial burst was followed by a sustained late phase production. the initial to KA within 30 min was in the cerebral suggesting that PG production at phase specific to hippocampus eicosanoids produced in the hippocampal further to were quantified for eicosanoids produced in the hippocampus, and As in 2 and amounts of and were to both in the initial phase and in the late phase, suggesting that and are to by 15-hydroxyprostaglandin dehydrogenase The levels of of PGD2 as with PGD2 were smaller of and suggesting that PGD2 PG and PGD2 also to which are M.H. 2005; PubMed Scopus Google Scholar), 1985; PubMed Scopus Google Scholar). The amounts of were the time suggesting are of PGD2 in the rat hippocampus of the PG by KA the initial phase hippocampal PG production was by KA receptors in the hippocampus, rats were with non-N-methyl-d-aspartate receptor selective for KA UBP301 selective for KA 30 min to the KA administration and UBP301 been as selective for KA receptors of and R. PubMed Scopus Google Scholar). As in of the PG productions were by and UBP301 were UBP296, inhibited production with When smaller amounts of KA (1 were the rats epileptic within by administration PG after intracerebroventricular KA administration were to by KA administration results suggest that burst PG productions in the hippocampus were by direct activation of KA receptors in the brain, by expressed in the of in the after Systemic KA the mechanism of KA-induced PG productions, we the levels of enzymes in the arachidonic acid cascade at mRNA and Reverse analysis of hippocampal tissues after KA an in the COX-2 and after KA were performed for cPLA2α, and COX-2 COX-2 mRNA increased a peak after KA cPLA2α and COX-1 analysis a low expression of COX-2 in rats, and a was after KA administration, which for The of cPLA2α These results that KA administration stimulates COX-2 up-regulation through KA the time course was from the initial burst PG production the COX-2 up-regulation was with the late phase PG suggesting a role of increased COX-2 in the late phase production. of COX on the PG further of the initial phase PG of COX were As in of rats with a inhibitor, NS398, the initial phase PG production. NS398 inhibited the production of PGD2 and TxB2 by whereas other PGs and were completely a COX inhibitor, inhibited of the PG productions. These results demonstrate that COX-2 constitutively present in the brain and a role in PG productions, and that COX-1 to in the production of PGD2 and In the present study, we profiled the eicosanoid production of rat hippocampal tissues during KA-induced seizure using the quantification method (13Kita Y. Takahashi T. Uozumi N. Nallan L. Gelb M.H. Shimizu T. Biochem. Biophys. Res. Commun. 2005; 330: 898-906Crossref PubMed Scopus (33) Google Scholar, 14Kita Y. Takahashi T. Uozumi N. Shimizu T. Anal. Biochem. 2005; 342: 134-143Crossref PubMed Scopus (107) Google Scholar). of the advantages of that eicosanoids the a for PG production in KA-induced seizure of in the KA administration caused PG production an burst of PG production within 30 min after KA administration. late phase PG production sustained to with levels the initial phase but the The productions of and PGD2 in the initial phase well with a (6Baran H. Heldt R. Hertting G. Brain Res. 1987; 404: 107-112Crossref PubMed Scopus (54) Google Scholar). In the of in that the COX in the hippocampus during K. Hickey R.W. Rose M.E. Zhu L. Chen J. Graham S.H. Brain Res. 2005; 1050: 130-137Crossref PubMed Scopus (86) Google Scholar) an increased production in the rat hippocampus after KA administration. demonstrate that and PGD2 are even after KA of the of PGs that and are to the in the hippocampus, whereas PGD2 to and with the of Biophys. 1994; PubMed Scopus Google Scholar, M. H. Lipid 2002; PubMed Scopus Google Scholar, J. 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Rose M.E. Zhu L. Chen J. Graham S.H. Brain Res. 2005; 1050: 130-137Crossref PubMed Scopus (86) Google Scholar). The COX-2 up-regulation in an increased production of but the levels of late phase productions were the initial due to the limited of arachidonic KA receptor signals stimulate arachidonic acid as in the initial phase The PG productions in the rats 1 and suggest in the hippocampus, a of arachidonic acid in the increased COX-2 levels with limited arachidonic acid may the of the late phase production In to the initial phase PG production that was specific to the hippocampus, we the late phase PG production also in the cortex has been that COX-2 up-regulation in the cortex as well as hippocampus (7Chen J. Marsh T. Zhang J.S. Graham S.H. Neuroreport. 1995; 6: 245-248Crossref PubMed Scopus (110) Google Scholar, 10Ciceri P. Zhang Y. Shaffer A.F. Leahy K.M. Woerner M.B. Smith W.G. Seibert K. Isakson P.C. J. Pharmacol. Exp. 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In such that KA directly stimulates KA are to hippocampus KA may stimulate through In of the PG the present study was to directly the of eicosanoids in epileptic seizure neuronal results an on such that are mechanisms of PG productions the initial phase and the late phase productions. are on the of COX in COX was to KA-induced seizure H. K. H. Brain Res. 1994; PubMed Scopus Google Scholar), and in COX E.J. E.J. Lee S.H. Moon Brain Res. 1999; PubMed Scopus Google Scholar, T. J. 2001; PubMed Scopus Google Scholar). of the studies were performed by using with which blocked the initial phase PG production. the periods of may on the inhibitor and the of administration, to the of the late phase production in KA-induced seizure by a with the initial phase and the late phase PG productions, administration method of well may a with which the initial phase A by Neuroscience. PubMed Scopus Google Scholar) that with the from KA-induced neuronal whereas the seizure suggesting the diversity of PG roles at the and at the with monitoring of PG amounts in understanding the of phase of PG production and the In the present study a dual phase regulatory mechanism of eicosanoid production in the rat hippocampus during KA-induced seizure. In to the temporal and results also the diversity of pathways to which COX-1 and COX-2 differentially A understanding of the mechanism of lipid mediator production a biochemical basis for understanding the roles of lipid mediators in studies lipid mediators in the brain Lipid 2005; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar) the of lipid mediators in excitotoxic using as a for understanding the roles of lipid mediators in the brain. We T. of Chiba, Japan) and M. M. Y. and K. University of Tokyo, Japan) for and